Field of the Invention
The present invention relates to a pharmaceutical composition containing an mRNA that is stabilised by sequence modifications in the translated region and is optimised for translation. The pharmaceutical composition according to the invention is suitable in particular as an inoculating agent and also as a therapeutic agent for tissue regeneration. Furthermore, a process for determining sequence modifications that stabilise mRNA and optimise mRNA translation is disclosed.
Description of the Prior Art
Gene therapy and genetic vaccination are tools of molecular medicine whose use in the treatment and prevention of diseases has considerable potential. Both of these approaches are based on the incorporation of nucleic acids into a patient's cells or tissue as well as on the subsequent processing of the information encoded by the incorporated nucleic acids, i.e. the expression of the desired polypeptides.
Conventional procedures involved in previous applications of gene therapy and genetic vaccination involved the use of DNA in order to incorporate the required genetic information into a cell. In this connection various processes for the incorporation of DNA into cells have been developed, such as for example calcium phosphate transfection, polyprene transfection, protoplast fusion, electroporation, microinjection and lipofection, in which connection lipofection in particular has proved to be a suitable process.
A further process that has been suggested in particular for the case of genetic vaccination involves the use of DNA viruses as DNA vehicles. Because such viruses are infectious, a very high transfection rate can be achieved when using DNA viruses as vehicles. The viruses used are genetically altered so that no functional infectious particles are formed in the transfected cell. Despite this precautionary measure, however, the risk of uncontrolled propagation of the introduced therapeutic gene as well as viral genes remains due to the possibility of recombination events.
Normally DNA incorporated into a cell is integrated to a certain extent into the genome of the transfected cell. On the one hand this phenomenon can exert a desirable effect, since in this way a long-lasting action of the introduced DNA can be achieved. On the other hand the integration into the genome brings with it a significant risk for gene therapy. Such integration events may, for example, involve an insertion of the incorporated DNA into an intact gene, which produces a mutation that interferes with or completely ablates the function of the endogenous gene. As a result of such integration events, enzyme systems that are important for cellular viability may be switched off. Alternatively, there is also the risk of inducing transformation of the transfected cell if the integration site occurs in a gene that is critical for regulating cell growth. Accordingly, when using DNA viruses as therapeutic agents and vaccines, a carcinogenic risk cannot be excluded. In this connection it should also be borne in mind that, in order to achieve effective expression of the genes incorporated into the cell, the corresponding DNA vehicles comprise a strong promoter, for example the viral CMV promoter. The integration of such promoters into the genome of the treated cell may, however, lead to undesirable changes in the regulation of the gene expression in the cell.
A further disadvantage of the use of DNA as a therapeutic agent or vaccine is the induction of pathogenic anti-DNA antibodies in the patient, resulting in a potentially fatal immune response.
In contrast to DNA, the use of RNA as a therapeutic agent or vaccine is regarded as significantly safer. In particular, use of RNA is not associated with a risk of stable integration into the genome of the transfected cell. In addition, no viral sequences such as promoters are necessary for effective transcription of RNA. Beyond this, RNA is degraded rapidly in vivo. Indeed, the relatively short half-life of RNA in circulating blood, as compared to that of DNA, reduces the risks associated with developing pathogenic anti-RNA antibodies. Indeed, anti-RNA antibodies have not been detected to date. For these reasons RNA may be regarded as the molecule of choice for molecular medicine therapeutic applications.
However, some basic problems still have to be solved before medical applications based on RNA expression systems can be widely employed. One of the problems in the use of RNA is the reliable, cell-specific and tissue-specific efficient transfer of the nucleic acid. Since RNA is normally found to be very unstable in solution, up to now RNA could not be used or used only very inefficiently as a therapeutic agent or inoculating agent in the conventional applications designed for DNA use.
Enzymes that break down RNA, so-called RNases (ribonucleases), are responsible in part for the instability. Even minute contamination by ribonucleases is sufficient to degrade down RNA completely in solution. Moreover, the natural decomposition of mRNA in the cytoplasm of cells is exquisitely regulated. Several mechanisms are known which contribute to this regulation. The terminal structure of a functional mRNA, for example, is of decisive importance. The so-called “cap structure” (a modified guanosine nucleotide) is located at the 5′ end and a sequence of up to 200 adenosine nucleotides (the so-called poly-A tail) is located at the 3′ end. The RNA is recognised as mRNA by virtue of these structures and these structures contribute to the regulatory machinery controlling mRNA degradation. In addition there are further mechanisms that stabilise or destabilise RNA. Many of these mechanisims are still unknown, although often an interaction between the RNA and proteins appears to be important in this regard. For example, an mRNA surveillance system has been described (Hellerin and Parker, Annu. Rev. Genet. 1999, 33: 229 to 260), in which incomplete or nonsense mRNA is recognised by specific feedback protein interactions in the cytosol and is made accessible to decomposition. Exonucleases appear to contribute in large measure to this process.
Certain measures have been proposed in the prior art to improve the stability of RNA and thereby enable its use as a therapeutic agent or RNA vaccine.
In EP-A-1083232 a process for the incorporation of RNA, in particular mRNA, into cells and organisms has been proposed in order to solve the aforementioned problem of the instability of RNA ex vivo. As described therein, the RNA is present in the form of a complex with a cationic peptide or protein.
WO 99/14346 describes further processes for stabilising mRNA. In particular, modifications of the mRNA are proposed that stabilise the mRNA species against decomposition by RNases. Such modifications may involve stabilisation by sequence modifications, in particular reduction of the C content and/or U content by base elimination or base substitution. Alternatively, chemical modifications may be used, in particular the use of nucleotide analogues, as well as 5′ and 3′ blocking groups, an increased length of the poly-A tail as well as the complexing of the mRNA with stabilising agents, and combinations of the aforementioned measures.
In US patents U.S. Pat. No. 5,580,859 and U.S. Pat. No. 6,214,804 mRNA vaccines and mRNA therapeutic agents are disclosed inter alia within the scope of “transient gene therapy” (TGT). Various measures are described therein for enhancing the translation efficiency and mRNA stability that relate in particular to the composition of the non-translated sequence regions.
Bieler and Wagner (in: Schleef (Ed.), Plasmids for Therapy and Vaccination, Chapter 9, pp. 147 to 168, Wiley-VCH, Weinheim, 2001) report on the use of synthetic genes in combination with gene therapy methods employing DNA vaccines and lentiviral vectors. The construction of a synthetic gag-gene derived from HIV-1 is described, in which the codons have been modified with respect to the wild type sequence (alternative codon usage) in such a way as to correspond to frequently used codons found in highly expressed mammalian genes. In this way, in particular, the A/T content compared to the wild type sequence was reduced. Moreover, the authors found an increased rate of expression of the synthetic gag gene in transfected cells. Furthermore, increased antibody formation against the gag protein was observed in mice immunised with the synthetic DNA construct. An increase in cytokine release in vitro from transfected spleen cells of such mice was also observed. Finally, an induction of a cytotoxic immune response in mice immunised with the gag expression plasmid was also found. The authors of this article attribute the improved properties of their DNA vaccine to a change in the nucleocytoplasmic transport of the mRNA expressed by the DNA vaccine, which was due to the optimised codon usage. The authors maintain that the effect of the altered codon usage on the translation efficiency was only slight.